PROJECT SUMMARY A recent finding from the Covert lab is that the majority of genes in the ?E. coli ?genome are predicted to be expressed less than once per generation, termed sub-generational expression. Sub-generational genes include many essential genes, as well as genes that are crucial for responding to different environmental challenges. Another feature of bacterial gene regulation is the operon, which is a genomic unit composed of several genes expressed under the control of a single promoter. This structural feature of the bacterial genome means that stochastic activation of one promoter can result in the simultaneous expression of multiple genes. Operons often contain functionally-related genes, and it has been hypothesized that operons exist to couple transcription and translation (though this has been long-debated in the field). In the context of sub-generational expression, this role of operon structure seems critical. For example, if two components of a heterodimer are both expressed sub-generationally from separate promoters it is unlikely that these two proteins will be present at the same time. However, even with a low probability of promoter activation, if the production of these two monomers is coupled through operon structure these transcriptional bursts will result in protein coexpression and functional heterodimers. Thus, the combination of sub-generational expression and operon structure could produce subpopulations of cells which simultaneously express all proteins in a complex or pathway, enabling these cells to respond to an environmental perturbation. Across a population of cells, these stochastic expression events create gene expression heterogeneity, which could protect the population against a large variety of potential challenges. Without operon structure, it is unlikely that these subpopulations of cells would exist. ?In this proposal, I will use both experimental and computational methods to determine the role of operon structure in the context of sub-generational expression, and evaluate if these two features of bacterial gene expression lead to increased fitness in response to environmental shifts?. Through this fellowship, I will 1) develop a novel understanding of the role of operon structure in ?E. coli?, 2) continue to develop skills to integrate computational and experimental approaches, and 3) develop my potential as an independent investigator. These goals will be made possible by the detailed research plan, my exceptionally qualified mentor, and the incredible facilities and training resources available at Stanford University.